The EGF receptor is a membrane glycoprotein with tyrosine-kinase activity. In general the result of EGF binding to its receptor is a mitogenic response. Epidermal growth factor has been shown to bind to osteoblastic cells in vivo and in vitro. In a bone organ culture system, the effects of EGF were increased bone resorption, increased PGE2 production and decreased collagen synthesis. EGF was also found to increase PGE2 production in osteosarcoma cell lines as well as primary culture cells from chick calvaria. In our laboratory we have shown that EGF increases cell proliferation in the G292 and SaoS cell lines and in primary osteoblasts isolated from neonatal rat calvaria. The mechanism by which tyrosine-kinase receptors produce their response in bone cells has not been characterized. It is the aim of our studies to continue the investigation of second messengers involved in the osteoblastic response to EGF. We have done studies using the tyrosine-kinase inhibitor, genistein, to examine the role of tyrosine phosphorylation in the EGF mitogenic response of G292 and primary culture cells. In the G292 cells the proliferation was inhibited by genistein, however in the primary culture cells proliferation was greatest in the cells treated with EGF and genistein. It is our goal to further explore the causes for the different response between the two cells types. We are planning to look at which proteins become tyrosine phosphorylated over a time course using Western transfer with an anti-phosphotyrosine antibody. In addition we will examine how genistein effects these tyrosine phosphorylations. In addition we are interested in the role of EGF on the electrophysiology of osteoblastic cells. There is evidence in the A431 human epidermoid carcinoma cell that EGF, in a tyrosine-kinase dependent fashion, can regulate Ca2+ channels. After characterizing the ion channels in our osteoblastic cells using patch-clamp techniques, we are planning to look at the role of EGF in regulation of ion channels. In summary, these studies will help to better understand the mechanisms of tyrosine-kinase action in osteoblastic cells.